Laser desorption/ionization method and mass spectrometry method

ABSTRACT

A laser desorption/ionization method, includes: a first step of preparing a sample support body including a substrate on which plurality of through holes opening to a first surface and a second surface facing each other are formed, and a conductive layer provided on at least the first surface; a second step of mounting a sample on a mounting surface of a mounting portion, and of disposing the sample support body on the sample such that the second surface is in contact with the sample; a third step of introducing a matrix solution into the plurality of through holes; and a fourth step of ionizing a component of the sample that is mixed with the matrix solution and is moved to the first surface side from the second surface side through the through hole by irradiating the first surface with laser light while a voltage is applied to the conductive layer.

TECHNICAL FIELD

The present disclosure relates to a laser desorption/ionization methodand a mass spectrometry method.

BACKGROUND ART

In the related art, a matrix-assisted laser desorption/ionization(MALDI) method has been known as a method of ionizing a sample such as abiological sample in order to perform mass spectrometry or the like. TheMALDI is a method of ionizing a sample by adding a low-molecular-weightorganic compound referred to as a matrix that absorbs laser light intothe sample, and by irradiating the sample with laser light. According tosuch a method, it is possible to ionize a thermally unstable substanceor a high-molecular-weight substance in a non-destructive manner(so-called soft ionization).

CITATION LIST Patent Literature

Patent Literature 1: U.S. Pat. No. 7,695,978

SUMMARY OF INVENTION Technical Problem

However, in the case of using the MALDI as described above in imagingmass spectrometry of imaging a two-dimensional distribution of moleculesconfiguring a sample, there is a limit to an increase in a resolution ofan image.

Therefore, an object of the present disclosure is to provide a laserdesorption/ionization method and a mass spectrometry method in which ahigh-molecular-weight sample can be ionized, and a resolution of animage in imaging mass spectrometry can be improved.

Solution to Problem

A laser desorption/ionization method of one aspect of the presentdisclosure, includes: a first step of preparing a sample support bodyincluding a substrate on which a plurality of through holes opening to afirst surface and a second surface facing each other are formed, and aconductive layer provided on at least the first surface; a second stepof mounting a sample on a mounting surface of a mounting portion, and ofdisposing the sample support body on the sample such that the secondsurface is in contact with the sample; a third step of introducing amatrix solution into the plurality of through holes; and a fourth stepof ionizing a component of the sample that is mixed with the matrixsolution and is moved to the first surface side from the second surfaceside through the through hole by irradiating the first surface withlaser light while a voltage is applied to the conductive layer, in astate in which the sample is disposed between the mounting portion andthe sample support body.

In the laser desorptionlionization method, the sample support body isdisposed on the sample, and the matrix solution is introduced into theplurality of through holes. The matrix solution is moved to the secondsurface side from the first surface side through each of the throughholes, and is mixed with the component of the sample. The component ofthe sample is mixed with the matrix solution and is moved to the firstsurface side from the second surface side through each of the throughholes. Then, in a case where the first surface is irradiated with thelaser light while a voltage is applied to the conductive layer, energyis transmitted to the component of the sample that is moved to the firstsurface side. Accordingly, the component of the sample is ionized. Inthe laser desorption/ionization method, the component of the sample isionized by being mixed with the matrix solution, and thus, it ispossible to reliably ionize a component of a high-molecular-weightsample. In addition, the component of the sample is moved to the firstsurface side through the plurality of through holes. For this reason, inthe component of the sample that is moved to the first surface side ofthe substrate, position information of the sample (two-dimensionaldistribution information of molecules configuring the sample) ismaintained. In such a state, the first surface of the substrate isirradiated with the laser light while a voltage is applied to theconductive layer, and thus, the component of the sample is ionized whilethe position information of the sample is maintained. Accordingly, it ispossible to improve a resolution of an image in imaging massspectrometry. As described above, according to the laserdesorption/ionization method, it is possible to ionize thehigh-molecular-weight sample and to improve the resolution of the imagein the imaging mass spectrometry.

A laser desorption/ionization method of one aspect of the presentdisclosure, includes: a first step of preparing a sample support bodyincluding a substrate on which a plurality of through holes opening to afirst surface and a second surface facing each other are formed, and aconductive layer provided on at least the first surface; a second stepof introducing a matrix solution into the plurality of through holes; athird step of mounting a sample on a mounting surface of a mountingportion, and of disposing the sample support body on the sample suchthat the second surface is in contact with the sample; and a fourth stepof ionizing a component of the sample that is mixed with the matrixsolution and is moved to the first surface side from the second surfaceside through the through hole by irradiating the first surface withlaser light while a voltage is applied to the conductive layer, in astate in which the sample is disposed between the mounting portion andthe sample support body.

In the laser desorption/ionization method, the sample support body inwhich the matrix solution is introduced into the plurality of throughholes is disposed on the sample. The component of the sample is mixedwith the matrix solution and is moved to the first surface side from thesecond surface side through each of the through holes. Then, in a casewhere the first surface is irradiated with the laser light while avoltage is applied to the conductive layer, energy is transmitted to thecomponent of the sample that is moved to the first surface side.Accordingly, the component of the sample is ionized. In the laserdesorption/ionization method, the component of the sample is ionized bybeing mixed with the matrix solution, and thus, it is possible toreliably ionize a component of a high-molecular-weight sample. Inaddition, the component of the sample is moved to the first surface sidethrough the plurality of through holes. For this reason, in thecomponent of the sample that is moved to the first surface side of thesubstrate, position information of the sample (two-dimensionaldistribution information of molecules configuring the sample) ismaintained. In such a state, the first surface of the substrate isirradiated with the laser light while a voltage is applied to theconductive layer, and thus, the component of the sample is ionized whilethe position information of the sample is maintained. Accordingly, it ispossible to improve a resolution of an image in imaging massspectrometry. As described above, according to the laserdesorption/ionization method, it is possible to ionize thehigh-molecular-weight sample and to improve the resolution of the imagein the imaging mass spectrometry.

In the laser desorption/ionization method of one aspect of the presentdisclosure, in the third step, the matrix solution may be dropped withrespect to the plurality of through holes from the first surface side.

In this case, it is possible to easily introduce the matrix solutioninto each of the through holes.

In the laser desorption/ionization method of one aspect of the presentdisclosure, in the second step, the matrix solution may be dropped withrespect to the plurality of through holes from the first surface side orthe second surface side. In this case, it is possible to easilyintroduce the matrix solution into each of the through holes.

In the laser desorption/ionization method of one aspect of the presentdisclosure, in the second step, the sample support body may be dipped inthe matrix solution. In this case, it is possible to easily introducethe matrix solution into each of the through holes.

In the laser desorption/ionization method of one aspect of the presentdisclosure, the sample may be a dried sample. In such a laserdesorption/ionization method, the component of the sample is mixed withthe matrix solution and is moved, and thus, even in a case where thesample is the dried sample, it is possible to smoothly move thecomponent of the sample.

A laser desorption/ionization method of one aspect of the presentdisclosure, includes: a first step of preparing a sample support bodyincluding a substrate having conductivity on which a plurality ofthrough holes opening to a first surface and a second surface facingeach other are formed; a second step of mounting a sample on a mountingsurface of a mounting portion, and of disposing the sample support bodyon the sample such that the second surface is in contact with thesample; a third step of introducing a matrix solution into the pluralityof through holes; and a fourth step of ionizing a component of thesample that is mixed with the matrix solution and is moved to the firstsurface side from the second surface side through the through hole byirradiating the first surface with laser light while a voltage isapplied to the substrate, in a state in which the sample is disposedbetween the mounting portion and the sample support body.

According to the laser desorption/ionization method, it is possible toomit the conductive layer from the sample support body, and to obtainthe same effect as that in the case of using the sample support bodyincluding the conductive layer as described above.

A laser desorption/ionization method of one aspect of the presentdisclosure, includes: a first step of preparing a sample support bodyincluding a substrate having conductivity on which a plurality ofthrough holes opening to a first surface and a second surface facingeach other are formed; a second step of introducing a matrix solutioninto the plurality of through holes; a third step of mounting a sampleon a mounting surface of a mounting portion, and of disposing the samplesupport body on the sample such that the second surface is in contactwith the sample; and a fourth step of ionizing a component of the samplethat is mixed with the matrix solution and is moved to the first surfaceside from the second surface side through the through hole byirradiating the first surface with laser light while a voltage isapplied to the substrate, in a state in which the sample is disposedbetween the mounting portion and the sample support body.

According to such a laser desorption/ionization method, it is possibleto omit the conductive layer from the sample support body, and to obtainthe same effect as that in the case of using the sample support bodyincluding the conductive layer as described above.

A mass spectrometry method of one aspect of the present disclosure,includes each of the steps of the laser desorption/ionization methoddescribed above; and a fifth step of detecting the component that isionized in the fourth step.

According to the mass spectrometry method, it is possible to performimaging mass spectrometry in which a high-molecular-weight sample can beionized, and a resolution of an image can be improved.

Advantageous Effects of Invention

According to the present disclosure, it is possible to provide a laserdesorption/ionization method and a mass spectrometry method in which ahigh-molecular-weight sample can be ionized, and a resolution of animage in imaging mass spectrometry can be improved.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a plan view of a sample support body that is used in a laserdesorption/ionization method and a mass spectrometry method of a firstembodiment.

FIG. 2 is a sectional view of the sample support body along line II-IIillustrated in FIG. 1.

FIG. 3 is a diagram illustrating an enlarged image of a substrate of thesample support body illustrated in FIG. 1.

FIG. 4 is a diagram illustrating steps of the mass spectrometry methodof the first embodiment.

FIG. 5 is a diagram illustrating the steps of the mass spectrometrymethod of the first embodiment.

FIG. 6 is a diagram illustrating the steps of the mass spectrometrymethod of the first embodiment.

(a) of FIG. 7 is a two-dimensional image according to a massspectrometry method of a comparative example, and (b) of FIG. 7 is amass spectrum according to the mass spectrometry method of thecomparative example.

(a) of FIG. 8 is a two-dimensional image according to a massspectrometry method of an example, and (b) of FIG. 8 is a mass spectrumaccording to the mass spectrometry method of the example.

(a) of FIG. 9 is a two-dimensional image according to a massspectrometry method of another example, and (b) of FIG. 9 is a massspectrum according to the mass spectrometry method of another example.

FIG. 10 is a diagram illustrating steps of a mass spectrometry method ofa second embodiment.

FIG. 11 is a diagram illustrating the steps of the mass spectrometrymethod of the second embodiment.

DESCRIPTION OF EMBODIMENTS

Hereinafter, embodiments of the present invention will be described indetail with reference to the drawings. Furthermore, in each of thedrawings, the same reference numerals will be applied to the sameportions or the corresponding portions, and the repeated descriptionwill be omitted.

First Embodiment

First, a sample support body that is used in a laserdesorption/ionization method and a mass spectrometry method of a firstembodiment and a second embodiment will be described. As illustrated inFIG. 1 and FIG. 2, a sample support body 1 includes a substrate 2, aframe 3, and a conductive layer 4. The substrate 2 includes a firstsurface 2 a and a second surface 2 b facing each other. A plurality ofthrough holes 2 c are formed on the substrate 2 uniformly (with ahomogeneous distribution). Each of the through holes 2 c extends along athickness direction of the substrate 2 (a direction perpendicular to thefirst surface 2 a and the second surface 2 b), and opens to the firstsurface 2 a and the second surface 2 b.

The substrate 2, for example, is formed of an insulating material intothe shape of a rectangular plate. The length of one side of thesubstrate 2 when seen from the thickness direction of the substrate 2,for example, is approximately several cm, and the thickness of thesubstrate 2, for example, is approximately 1 μm to 50 μm. The throughhole 2 c, for example, is approximately in the shape of a circle whenseen from the thickness direction of the substrate 2. The width of thethrough hole 2 c is 1 nm to 700 nm. The width of the through hole 2 cindicates the diameter of the through hole 2 c in a case where thethrough hole 2 c is approximately in the shape of a circle when seenfrom the thickness direction of the substrate 2, and indicates thediameter (an effective diameter) of a virtual maximum cylinder fallinginto the through hole 2 c in a case where the through hole 2 c is notapproximately in the shape of a circle. A pitch between the respectivethrough holes 2 c is 1 nm to 1000 nm. The pitch between the respectivethrough holes 2 c indicates a center-to-center distance of therespective circles in a case where the through hole 2 c is approximatelyin the shape of a circle when seen from the thickness direction of thesubstrate 2, and indicates a center axis-to-center axis distance of thevirtual maximum cylinder falling into the through hole 2 c in a casewhere the through hole 2 c is not approximately in the shape of acircle.

The frame 3 is provided on the first surface 2 a of the substrate 2.Specifically, the frame 3 is fixed to the first surface 2 a of thesubstrate 2 by an adhesive layer 5. It is preferable that an adhesivematerial having less emitted gas (for example, glass with a low meltingpoint, a vacuum adhesive agent, and the like) is used as the material ofthe adhesive layer 5. The frame 3 has approximately the same outer shapeas that of the substrate 2 when seen from the thickness direction of thesubstrate 2. An opening 3 a is formed in the frame 3. A portioncorresponding to the opening 3 a in the substrate 2 functions as aneffective region R for moving a component of a sample described below tothe first surface 2 a side.

The frame 3, for example, is formed into the shape of a rectangularplate by the insulating material. The length of one side of the frame 3when seen from the thickness direction of the substrate 2, for example,is approximately several cm, and the thickness of the frame 3, forexample, is less than or equal to 1 mm. The opening 3 a, for example, isin the shape of a circle when seen from the thickness direction of thesubstrate 2, and in such a case, the diameter of the opening 3 a, forexample, is approximately several mm to several tens of mm. By such aframe 3, the handling of the sample support body 1 is facilitated, andthe deformation of the substrate 2 due to a temperature change or thelike is suppressed.

The conductive layer 4 is provided on the first surface 2 a of thesubstrate 2. Specifically, the conductive layer 4 is formed in a regioncorresponding to the opening 3 a of the frame 3 on the first surface 2 aof the substrate 2 (that is, a region corresponding to the effectiveregion R), and is continuously (integrally) formed on an inner surfaceof the opening 3 a, and a surface 3 b on a side opposite to thesubstrate 2 of the frame 3. In the effective region R, the conductivelayer 4 covers a portion in which the through hole 2 c is not formed onthe first surface 2 a of the substrate 2. That is, in the effectiveregion R, each of the through holes 2 c is exposed to the opening 3 a.

The conductive layer 4 is formed of a conductive material. However, itis preferable that a metal having low affinity (reactivity) with respectto a sample S and high conductivity is used as the material of theconductive layer 4, from the following reasons.

For example, in a case where the conductive layer 4 is formed of a metalsuch as copper (Cu) having high affinity with respect to a sample suchas protein, in a process of ionizing the sample described below, thesample is ionized in a state where Cu atoms are attached to samplemolecules. Then, there is a concern that a detection result is shiftedin the mass spectrometry method described below as the Cu atoms areattached. Therefore, it is preferable that a metal having low affinitywith respect to the sample is used as the material of the conductivelayer 4.

On the other hand, a metal easily and stably applies a constant voltageas conductivity is high. For this reason, in a case where the conductivelayer 4 is formed of a metal having high conductivity, it is possible tohomogeneously apply a voltage to the first surface 2 a of the substrate2 in the effective region R. In addition, there is a tendency that ametal has high thermal conductivity as conductivity is high. For thisreason, in a case where the conductive layer 4 is formed of a metalhaving high conductivity, it is possible to efficiently transfer theenergy of laser light that is applied to the substrate 2 to the samplethrough the conductive layer 4. Therefore, it is preferable that a metalhaving high conductivity is used as the material of the conductive layer4.

From the viewpoint described above, for example, it is preferable thatgold (Au), platinum (Pt), and the like are used as the material of theconductive layer 4. The conductive layer 4, for example, is formed tohave a thickness of approximately 1 nm to 350 nm by a plating method, anatomic layer deposition (ALD) method, a vapor deposition method, asputtering method, and the like. Furthermore, for example, chromium(Cr), nickel (Ni), titanium (Ti), and the like may be used as thematerial of the conductive layer 4.

FIG. 3 is a diagram illustrating an enlarged image of the substrate 2when seen from the thickness direction of the substrate 2. In FIG. 3, ablack portion is the through hole 2 c, and a white portion is apartition portion between the through holes 2 c. As illustrated in FIG.3, the plurality of through holes 2 c having an approximately constantwidth are uniformly formed on the substrate 2. It is preferable that anopening rate of the through holes 2 c in the effective region R (a ratioof all of the through holes 2 c to the effective region R when seen fromthe thickness direction of the substrate 2) is practically 10% to 80%,and is particularly 60% to 80%. The sizes of the plurality of throughholes 2 c may be uneven with each other, and the plurality of throughholes 2 c may be partially connected to each other.

The substrate 2 illustrated in FIG. 3 is an alumina porous film that isformed by performing anodic oxidation with respect to aluminum (Al).Specifically, an anodic oxidation treatment is performed with respect toan Al substrate, and a surface portion that is oxidized is peeled offfrom the Al substrate, and thus, it is possible to obtain the substrate2. Furthermore, the substrate 2 may be formed by performing anodicoxidation with respect to a valve metal other than Al, such as tantalum(Ta), niobium (Nb), titanium (Ti), hafnium (Hf), zirconium (Zr), zinc(Zn), tungsten (W), bismuth (Bi), and antimony (Sb), or may be formed byperforming anodic oxidation with respect to silicon (Si).

Next, the laser desorption/ionization method and the mass spectrometrymethod of the first embodiment in which the sample support body 1 isused will be described. In FIG. 4 to FIG. 6, the through hole 2 c, theconductive layer 4, and the adhesive layer 5 in the sample support body1 are not illustrated. In addition, for the convenience of illustration,a dimensional ratio or the like is different between the sample supportbody 1 illustrated in FIG. 1 and FIG. 2 and the sample support body 1illustrated in FIG. 4 to FIG. 6.

First, the sample support body 1 described above is prepared (a firststep). The sample support body 1 may be prepared by being produced by aperson who carries out the laser desorption/ionization method and themass spectrometry method, or may be prepared by being acquired from aproducer, a seller, or the like of the sample support body 1.

Subsequently, as illustrated in FIG. 4(a), the sample S that is a massspectrometry target is mounted on a mounting surface 6 a of a glassslide (a mounting portion) 6 (a second step). The glass slide 6 is aglass substrate on which a transparent conductive film such as an indiumtin oxide (ITO) film is formed, and in the glass slide 6, the surface ofthe transparent conductive film is the mounting surface 6 a.Furthermore, not only the glass slide 6 but also a member that iscapable of ensuring conductivity (for example, a substrate or the likefowled of a metal material, such as stainless steel, or the like) can beused as the mounting portion. Subsequently, as illustrated in (b) ofFIG. 4, the sample support body 1 is disposed on the sample S such thatthe second surface 2 b is in contact with the sample S (the secondstep). At this time, the sample S is disposed in the effective region Rwhen seen from the thickness direction of the substrate 2. Here, thesample S, for example, is a thin film-like biological sample such as atissue slice. The sample S is a dried sample. In addition, in order tosmoothly move a component S1 of the sample S (refer to FIG. 6), asolution for decreasing the viscosity of the component S1 (for example,an acetonitrile mixed liquid or the like) may be mixed with the sampleS. Subsequently, as illustrated in (a) of FIG. 5, the sample supportbody 1 is fixed to the glass slide 6, in a state where the secondsurface 2 b of the substrate 2 is in contact with the sample S. At thistime, the sample support body I is fixed to the glass slide 6 by a tape7 having conductivity (for example, a carbon tape or the like).

Subsequently, as illustrated in (b) of FIG. 5, a matrix solution 81 isintroduced into the plurality of through holes 2 c of the sample supportbody 1 (refer to FIG. 2) (a third step). Specifically, the matrixsolution 81, for example, is dropped with respect to the plurality ofthrough holes 2 c from the first surface 2 a side of the sample supportbody 1 by a pipette 8. The matrix solution 81 is dropped intoapproximately the entire region of the effective region R to reach theentire region of the sample S. More preferably, the matrix solution 81,for example, is applied with respect to the plurality of through holes 2c from the first surface 2 a side of the sample support body 1 withapproximately a uniform amount by an air brush or the like. The matrixsolution 81 is moved towards the second surface 2 b side from the firstsurface 2 a side of the sample support body 1 through each of thethrough holes 2 c. Then, the matrix solution 81 is mixed with the sampleS that is in contact with the second surface 2 b of the sample supportbody 1 in each of the through holes 2 c. The matrix solution 81 may beprepared by being produced by a person who carries out the laserdesorption/ionization method and the mass spectrometry method, or may beprepared by being acquired from a producer, a seller, or the like of thematrix solution 81.

The matrix solution 81 is a solution containing a matrix. The matrixsolution 81, for example, is a solution that is prepared by dissolving10 mg of a matrix in 1 ml of acetonitrile, or the like. The matrix is anorganic compound that absorbs laser light. The matrix, for example, is a2,5-dihydroxybenzoic acid (DHB) or the like.

As illustrated in (a) of FIG. 6, in each of the through holes 2 c, thecomponent S1 of the sample S is mixed with the matrix solution 81 thatis moved to the second surface 2 b side of the sample support body 1,and is moved towards the first surface 2 a side from the second surface2 b side of the sample support body 1 through each of the through holes2 c. Then, a mixture S2 of the component S1 and the matrix solution 81is accumulated on the first surface 2 a side of the sample support body1 in each of the through holes 2 c by a surface tension.

Subsequently, as illustrated in (b) of FIG. 6, the glass slide 6, thesample support body 1, and the sample S are mounted on a support portion12 of a mass spectrometry device 10 (for example, a stage), in a statewhere the sample S is disposed between the glass slide 6 and the samplesupport body 1. Subsequently, a voltage is applied to the conductivelayer 4 of the sample support body 1 (refer to FIG. 2) through themounting surface 6 a of the glass slide 6 and the tape 7 by a voltageapplication unit 14 of the mass spectrometry device 10 (a fourth step).Subsequently, the first surface 2 a of the substrate 2 is irradiatedwith laser light L through the opening 3 a of the frame 3 by a laserlight irradiation unit 13 of the mass spectrometry device 10 (the fourthstep).

That is, the laser light L is applied to a region corresponding to theopening 3 a of the frame 3 on the first surface 2 a of the substrate 2(that is, a region corresponding to the effective region R). Here, thelaser light irradiation unit 13 scans the region corresponding to theeffective region R with the laser light L. Furthermore, the scanning ofthe laser light L with respect to the region corresponding to theeffective region R can be carried out by operating at least one of thesupport portion 12 and the laser light irradiation unit 13.

As described above, the first surface 2 a of the substrate 2 isirradiated with the laser light L while a voltage is applied to theconductive layer 4, the component S1 that is moved to the first surface2 a side of the substrate 2 is ionized, and a sample ion S3 (thecomponent S1 that is ionized) is emitted (the fourth step).Specifically, the conductive layer 4 (refer to FIG. 2), and the matrixin the matrix solution 81 that is moved to the first surface 2 a side ofthe substrate 2 along with the component S1 absorb the energy of thelaser light L. The matrix is gasified along with the component S1 by theenergy. Then, a proton or a cation is added to the molecules of thecomponent S1 that is gasified, and thus, the gasified component S1becomes the sample ion S3. The first step to the fourth step describedabove correspond to the laser desorption/ionization method using thesample support body 1.

The sample ion S3 that is emitted is moved towards a ground electrode(not illustrated) that is provided between the sample support body 1 andan ion detection unit 15 while being accelerated. That is, the sampleion S3 is moved towards the ground electrode while being accelerated bya potential difference that occurs between the conductive layer 4 towhich a voltage is applied and the ground electrode. Then, the sampleion S3 is detected by the ion detection unit 15 of the mass spectrometrydevice 10 (a fifth step). Here, the ion detection unit 15 detects thesample ion S3 to correspond to a scanning position of the laser light L.Accordingly, it is possible to image a two-dimensional distribution ofthe molecules configuring the sample S. Furthermore, here, the massspectrometry device 10 is a scanning mass spectrometry device using atime-of-flight mass spectrometry (TOF-MS) method. The first step to thefifth step described above correspond to the mass spectrometry methodusing the sample support body 1.

As described above, in the laser desorption/ionization method of thefirst embodiment, the sample support body 1 is disposed on the sample S,and the matrix solution 81 is introduced into the plurality of throughholes 2 c. The matrix solution 81 is moved to the second surface 2 bside from the first surface 2 a side through each of the through holes 2c, and is mixed with the component S1 of the sample S. The component S1is mixed with the matrix solution 81, and is moved to the first surface2 a side from the second surface 2 b side through each of the throughholes 2 c. Then, in a case where the first surface 2 a is irradiatedwith the laser light L while a voltage is applied to the conductivelayer 4, the energy is transmitted to the component S1 that is moved tothe first surface 2 a side. Accordingly, the component S1 is ionized. Inthe laser desorption/ionization method, the component S1 is ionized bybeing mixed with the matrix solution 81, and thus, it is possible toreliably ionize the component S1 of the high-molecular-weight sample S.In addition, the component S1 is moved to the first surface 2 a sidethrough the plurality of through holes 2 c. For this reason, in thecomponent S1 that is moved to the first surface 2 a side of thesubstrate 2, position information of the sample S (two-dimensionaldistribution information of the molecules configuring the sample S) ismaintained. In such a state, the first surface 2 a of the substrate 2 isirradiated with the laser light L while a voltage is applied to theconductive layer 4, and thus, the component S1 of the sample S isionized while the position information of the sample S is maintained.Accordingly, it is possible to improve a resolution of an image inimaging mass spectrometry. As described above, according to the laserdesorption/ionization method, it is possible to ionize thehigh-molecular-weight sample S and to improve the resolution of theimage in the imaging mass spectrometry.

In addition, in the laser desorption/ionization method of the firstembodiment, in the third step, the matrix solution 81 is dropped withrespect to the plurality of through holes 2 c from the first surface 2 aside. In this case, it is possible to easily introduce the matrixsolution 81 to each of the through holes 2 c.

In addition, in the laser desorption/ionization method of the firstembodiment, the sample S is the dried sample. In the laserdesorption/ionization method, the component S1 of the sample S is mixedwith the matrix solution 81 and is moved, and thus, even in a case wherethe sample S is the dried sample, it is possible to smoothly move thecomponent S1.

According to the mass spectrometry method of the first embodiment, it ispossible to perform imaging mass spectrometry in which thehigh-molecular-weight sample S can be ionized, and the resolution of theimage can be improved.

FIG. 7 is a diagram illustrating a result according to a massspectrometry method of a comparative example. In the comparativeexample, the sample support body 1 was used, but the matrix solution 81was not used, and a two-dimensional distribution of a molecular weightof a dried brain slice of a mouse (the sample S) was imaged. Asillustrated in (a) of FIG. 7, in the mass spectrometry method of thecomparative example, it was not possible to obtain an ion image ofphosphatide. Furthermore, in (a) of FIG. 7, an outline L1 of the sampleS is represented by a virtual line. In addition, as illustrated in (b)of FIG. 7, in the mass spectrometry method of the comparative example,it was not possible to obtain a signal of the phosphatide.

FIG. 8 is a diagram illustrating a result according to a massspectrometry method of an example. In the example, the sample supportbody I and the matrix solution 81 (a DHB matrix) were used, and as withthe comparative example, a two-dimensional distribution of a molecularweight of a dried brain slice of a mouse (the sample S) was imaged. Asillustrated in (a) of FIG. 8, in the mass spectrometry method of theexample, it was possible to obtain an ion image of phosphatide.Furthermore, in (a) of FIG. 8, an outline L2 of the sample S isrepresented by a virtual line. In addition, as illustrated in (b) ofFIG. 8, in the mass spectrometry method of the example, it was possibleto obtain a signal of the phosphatide.

FIG. 9 is a diagram illustrating a result according to a massspectrometry method of another example. In another example, the samplesupport body 1 and the matrix solution 81 (the DHB matrix) were used,and a two-dimensional distribution of a molecular weight of a driedliver slice of a mouse (the sample S) was imaged. As illustrated in (a)of FIG. 9, in the mass spectrometry method of another example, it waspossible to obtain an ion image of phosphatide. Furthermore, in (a) ofFIG. 9, an outline L3 of the sample S is represented by a virtual line.In addition, as illustrated in (b) of FIG. 9, in the mass spectrometrymethod of the example, it was possible to obtain a signal of thephosphatide.

In addition, in the mass spectrometry method of the example and anotherexample, the width of each of the through holes 2 c was 1 nm to 700 nm,and thus, it was possible to obtain sufficient signal intensity withrespect to a high-molecular-weight sample.

Second Embodiment

Next, a laser desorption/ionization method and a mass spectrometrymethod of a second embodiment in which the sample support body 1 is usedwill be described. The laser desorption/ionization method and the massspectrometry method of the second embodiment are mainly different fromthe laser desorption/ionization method and the mass spectrometry methodof the first embodiment in that the matrix solution 81 is introducedinto the through hole 2 c of the sample support body 1, and then, thesample support body 1 into which the matrix solution 81 is introduced isdisposed on the sample S. That is, in the laser desorption/ionizationmethod and the mass spectrometry method of the second embodiment, theorders of the second step and the third step of the laserdesorption/ionization method and the mass spectrometry method of thefirst embodiment are changed. In the laser desorption/ionization methodand the mass spectrometry method of the second embodiment, the othersare the same as the laser desorption/ionization method and the massspectrometry method of the first embodiment, and thus, the detaileddescription will be omitted. In FIG. 10 and FIG. 11, the through hole 2c, the conductive layer 4, and the adhesive layer 5 in the samplesupport body 1 are not illustrated. In addition, for the convenience ofillustration, a dimensional ratio or the like is different between thesample support body I illustrated in FIG. 1 and FIG. 2 and the samplesupport body 1 illustrated in FIG. 10 and FIG. 11.

First, as illustrated in (a) of FIG. 10, the sample support body 1described above is prepared (a first step). Subsequently, the matrixsolution 81 is introduced into the plurality of through holes 2 c of thesample support body I (refer to FIG. 2) (a second step). Specifically,the matrix solution 81, for example, is dropped with respect to theplurality of through holes 2 c from the first surface 2 a of the samplesupport body 1 by the pipette 8. The matrix solution 81 is dropped intoapproximately the entire region of the effective region R. Morepreferably, the matrix solution 81, for example, is applied with respectto the plurality of through holes 2 c from the first surface 2 a side ofthe sample support body 1 with approximately a uniform amount by an airbrush or the like. The matrix solution 81 is moved towards the secondsurface 2 b side from the first surface 2 a side of the sample supportbody 1 through each of the through holes 2 c. Each of the through holes2 c is filled with the matrix solution 81.

Subsequently, as illustrated in (b) of FIG. 10, the sample S is mountedon the mounting surface 6 a of the glass slide 6 (a third step).Subsequently, as illustrated in (a) of FIG. 11, the sample support body1 is disposed on the sample S such that the second surface 2 b is incontact with the sample S (the third step). Subsequently, as illustratedin (b) of FIG. 11, as with the first embodiment, the sample support body1 is fixed to the glass slide 6 by the tape 7. The matrix solution 81 ineach of the through holes 2 c is mixed with the sample S that is incontact with the second surface 2 b of the sample support body 1 in eachof the through holes 2 c. In each of the through holes 2 c, thecomponent S of the sample S is mixed with the matrix solution 81, and ismoved towards the first surface 2 a side from the second surface 2 bside of the sample support body 1 through each of the through holes 2 c.Then, a mixture S2 of the component S1 and the matrix solution 81 isaccumulated on the first surface 2 a side of the sample support body 1in each of the through holes 2 c by a surface tension.

Subsequently, as with the first embodiment (refer to (b) of FIG. 6), thefirst surface 2 a of the sample support body 1 is irradiated with thelaser light L by the laser light irradiation unit 13 while a voltage isapplied to the conductive layer 4 (refer to FIG. 2) by the voltageapplication unit 14, in a state where the sample S is disposed betweenthe glass slide 6 and the sample support body 1. Accordingly, thecomponent S1 that is moved to the first surface 2 a side of thesubstrate 2 is ionized, and the sample ion S3 (the component S1 that isionized) is emitted (a fourth step).

Then, as with the first embodiment, the sample ion S3 is detected by theion detection unit 15 of the mass spectrometry device 10 (a fifth step).Furthermore, the laser desorption/ionization method of the secondembodiment includes each of the steps from the first step to the fourthstep described above. The mass spectrometry method of the secondembodiment includes each of the steps from the first step to the fifthstep described above.

As described above, in the laser desorption/ionization method of thesecond embodiment, the sample support body 1 in which the matrixsolution 81 is introduced into the plurality of through holes 2 c isdisposed on the sample S. The component S1 of the sample S is mixed withthe matrix solution 81, and is moved to the first surface 2 a side fromthe second surface 2 b side through each of the through holes 2 c.According to the laser desorption/ionization method of the secondembodiment, as with the laser desorption/ionization method of the firstembodiment described above, it is possible to ionize thehigh-molecular-weight sample S and to improve a resolution of an imagein imaging mass spectrometry.

In addition, in the laser desorption/ionization method of the secondembodiment, in the second step, the matrix solution 81 is dropped withrespect to the plurality of through holes 2 c from the first surface 2 aside. In this case, it is possible to easily introduce the matrixsolution 81 into each of the through holes 2 c.

The present disclosure is not limited to the embodiments describedabove. In each of the embodiments, for example, the conductive layer 4may not be provided on the second surface 2 b of the substrate 2 and theinner surface of the through hole 2 c insofar as the conductive layer 4is provided on at least first surface 2 a of the substrate 2. Inaddition, the conductive layer 4 may be provided on the second surface 2b of the substrate 2 and the inner surface of the through hole 2 c. Inaddition, the sample support body 1 may be fixed to the glass slide 6 bymeans other than the tape 7 (for example, means using an adhesive agent,a fixing tool, or the like).

In addition, in the fourth step of each of the embodiments, a voltagemay be applied to the conductive layer 4 without the mounting surface 6a of the glass slide 6 and the tape 7. In this case, the glass slide 6and the tape 7 may not have conductivity. In addition, the substrate 2may have conductivity, and in the fourth step, a voltage may be appliedto the substrate 2. In this case, it is possible to omit the conductivelayer 4 from the sample support body 1, and to obtain the same effect asthat in the case of using the sample support body 1 including theconductive layer 4 as described above.

In addition, in each of the embodiments, in the mass spectrometry device10, the region corresponding to the effective region R may be irradiatedwith the laser light L by the laser light irradiation unit 13 at onetime, and the sample ion S3 may be detected by the ion detection unit 15while two-dimensional information of the region is maintained. That is,the mass spectrometry device 10 may be a stigmatic mass spectrometrydevice.

In addition, the laser desorption/ionization method of each of theembodiments described above can be used not only in the imaging massspectrometry in which the two-dimensional distribution of the moleculesconfiguring the sample S is imaged, but also in other measurements andtests such as ion mobility measurement.

In addition, in the second embodiment, an example has been described inwhich in the second step, the matrix solution 81 is dropped with respectto the plurality of through holes 2 c from the first surface 2 a, butthe matrix solution 81 may be dropped with respect to the plurality ofthrough holes 2 c from the second surface 2 b. In addition, in thesecond embodiment, in the second step, the sample support body 1 may bedipped in the matrix solution 81. In any case, it is possible to easilyintroduce the matrix solution 81 to each of the through holes 2 c.

In addition, in each of the embodiments, an example has been describedin which the opening 3 a of the frame 3 is in the shape of a circle whenseen from the thickness direction of the substrate 2, but the opening 3a may be in various shapes. The opening 3 a of the frame 3, for example,may be in the shape of a rectangle.

In addition, in each of the embodiments, an example has been describedin which the sample S is the dried sample, but the sample S may be anaqueous sample.

In addition, in each of the embodiments, an example has been describedin which the sample S is mounted on the glass slide 6, but the sample Smay be directly mounted on the support portion 12 of the massspectrometry device 10. At this time, the support portion 12 of the massspectrometry device 10 corresponds to the glass slide 6. Reference SignsList

1: sample support body, 2: substrate, 2 a: first surface, 2 b: secondsurface, 2 c: through hole, 4: conductive layer, 6: glass slide(mounting portion), 6 a: mounting surface, 81: matrix solution, L: laserlight, S: sample, S1: component.

1: A laser desorption/ionization method, comprising: a first step ofpreparing a sample support body including a substrate on which aplurality of through holes opening to a first surface and a secondsurface facing each other are formed, and a conductive layer provided onat least the first surface; a second step of mounting a sample on amounting surface of a mounting portion, and of disposing the samplesupport body on the sample such that the second surface is in contactwith the sample; a third step of introducing a matrix solution into theplurality of through holes; and a fourth step of ionizing a component ofthe sample that is mixed with the matrix solution and is moved to thefirst surface side from the second surface side through the through holeby irradiating the first surface with laser light while a voltage isapplied to the conductive layer, in a state in which the sample isdisposed between the mounting portion and the sample support body. 2: Alaser desorption/ionization method, comprising: a first step ofpreparing a sample support body including a substrate on which aplurality of through holes opening to a first surface and a secondsurface facing each other are formed, and a conductive layer provided onat least the first surface; a second step of introducing a matrixsolution into the plurality of through holes; a third step of mounting asample on a mounting surface of a mounting portion, and of disposing thesample support body on the sample such that the second surface is incontact with the sample; and a fourth step of ionizing a component ofthe sample that is mixed with the matrix solution and is moved to thefirst surface side from the second surface side through the through holeby irradiating the first surface with laser light while a voltage isapplied to the conductive layer, in a state in which the sample isdisposed between the mounting portion and the sample support body. 3:The laser desorption/ionization method according to claim 1, wherein inthe third step, the matrix solution is dropped with respect to theplurality of through holes from the first surface side. 4: The laserdesorption/ionization method according to claim 2, wherein in the secondstep, the matrix solution is dropped with respect to the plurality ofthrough holes from the first surface side or the second surface side. 5:The laser desorption/ionization method according to claim 2, wherein inthe second step, the sample support body is dipped in the matrixsolution. 6: The laser desorption/ionization method according to claim1, wherein the sample is a dried sample. 7: A laserdesorption/ionization method, comprising: a first step of preparing asample support body including a substrate having conductivity on which aplurality of through holes opening to a first surface and a secondsurface facing each other are formed; a second step of mounting a sampleon a mounting surface of a mounting portion, and of disposing the samplesupport body on the sample such that the second surface is in contactwith the sample; a third step of introducing a matrix solution into theplurality of through holes; and a fourth step of ionizing a component ofthe sample that is mixed with the matrix solution and is moved to thefirst surface side from the second surface side through the through holeby irradiating the first surface with laser light while a voltage isapplied to the substrate, in a state in which the sample is disposedbetween the mounting portion and the sample support body. 8: A laserdesorption/ionization method, comprising: a first step of preparing asample support body including a substrate having conductivity on which aplurality of through holes opening to a first surface and a secondsurface facing each other are formed; a second step of introducing amatrix solution into the plurality of through holes; a third step ofmounting a sample on a mounting surface of a mounting portion, and ofdisposing the sample support body on the sample such that the secondsurface is in contact with the sample; and a fourth step of ionizing acomponent of the sample that is mixed with the matrix solution and ismoved to the first surface side from the second surface side through thethrough hole by irradiating the first surface with laser light while avoltage is applied to the substrate, in a state in which the sample isdisposed between the mounting portion and the sample support body. 9: Amass spectrometry method, comprising: each of the steps of the laserdesorption/ionization method according to claim 1; and a fifth step ofdetecting the component that is ionized in the fourth step.
 10. Thelaser desorption/ionization method according to claim 2, wherein thesample is a dried sample.
 11. A mass spectrometry method, comprising:each of the steps of the laser desorption/ionization method according toclaim 2; and a fifth step of detecting the component that is ionized inthe fourth step.
 12. A mass spectrometry method, comprising: each of thesteps of the laser desorption/ionization method according to claim 7;and a fifth step of detecting the component that is ionized in thefourth step.
 13. A mass spectrometry method, comprising: each of thesteps of the laser desorption/ionization method according to claim 8;and a fifth step of detecting the component that is ionized in thefourth step.